Financial support: This work was funded by the
Consejo Nacional de Ciencia y Tecnología (Project P48831).

Keywords: cocultivation, GUS assay.

Abstract

Most of the pepper species of
the genus Capsicum have been recalcitrant to efficient Agrobacterium tumefaciens-mediated stable
or transient, genetic transformation. In the present work, we optimized
a protocol for transient transformation of the Habanero pepper (Capsicum chinense Jacq.) through the
standardization of several experimental factors. These included the age of the
plants, the temperature, the length of co-cultivation, the application of a
negative (vacuum) and/or a positive (infiltration) pressure, along with micro injection,
the use of acetosyringone during the bacterial culturing, and modification of
the pH during the GUS assay to eliminate the endogenous β-glucuronidase
activity. The standardized protocol, which yielded nearly 55% fully
transformed leaf explants, was used to successfully mobilize two empty binary
vectors (pCAMBIA2301 and pCAMex), as well as the C. chinense cDNAs
encoding the pathogenesis-related protein 10 and esterase, respectively.

Transient
genetic transformation could be a valuable tool and offer a rapid assessment of
gene function prior to stable transformation. By using transient genetic
transformation, which is a relatively fast process that would require only 5-8
days from infiltration to gene expression, transformation may occur at high
frequencies (Fischer et al. 1999; Van der Hoorn et al. 2000) and as result a
large number of constructs may be tested (Kapila et al. 1997). In recent studies, Santana-Buzzy et al. (2005) and
Zapata-Castillo et al. (2007) were able to regenerate shoots from the
leaf-derived callus of the Habanero pepper. Thus, the transient transformation
of Habanero pepper explants might also be exploited to attempt the stable
transformation of pepper via organogenesis. In the present study, we assessed
several factors to optimize an A. tumefaciens-mediated transient transformation
protocol for Habanero pepper leaf explants. The protocol was successfully used
to mobilize the full-cDNAs of the genes encoding the pathogenesis-related
protein 10 (EMBL AJ879115) and esterase (EMBL AJ879119), which were cloned from
a C. chinense cDNA library constructed expressly from Phytophthora
capsici-elicited cell suspensions.

Materials and Methods

Biological materials and binary vectors

Aseptically produced Habanero pepper
plantlets of the “naranja” variety (Seminis®) were used as the source of
explants. The pepper seeds were surface sterilized by immersion in 70% (v/v)
ethanol for 5 min, rinsed twice in sterile distilled water and again in a 30%
(v/v) solution of commercial chlorine (Cloralex®, 6% active chlorine) for 15
min with constant stirring. Then the seeds were thoroughly rinsed four times
with sterile distilled water, and placed on sterile filter paper to eliminate
an excess of water. The seeds were then preconditioned by spending five days at
25 ± 2ºC, in darkness, on Petri dishes of MS medium (Murashige and Skoog, 1962)
supplemented with 3% sucrose, 0.2% (v/v) of Gelrite®, without growth regulators
and at a pH of 5.6. Finally, the seeds were transferred to a 16 hrs photoperiod
(photon flux density of 40-60 µmol·m-2·s-1, provided by
39W fluorescent lamps, Phillips, USA) at 25 ± 2ºC. Upon completion of
germination, the plantlets were transferred to magenta containers to wait until
they were ready for transformation.

Agrobacterium tumefaciens strain LBA4404, either disarmed or
transformed with empty or recombinant binary vectors (pCAMBIA2301/pCAMex
or pCAMex::PR10/pCAMex::Esterase,
respectively) were used for co-cultivation. The pCAMex binary vector was
constructed by mobilizing the 35S promoter and the OCS terminator sequences,
derived from the pART7 plasmid (Gleave, 1992), into the multiple cloning site
of the pCAMBIA1303 binary vector (http://www.cambia.org.au)
(Figure 1). pCAMex::PR10 and pCAMex::Esterase were constructed by sub-cloning the
corresponding full complementary DNAs encoding the pathogenesis-related 10
(PR10) and esterase proteins, from pTriplEx2 (Clontech, Palo Alto, Cal.), into
the pCAMex plasmid digested with Kpn I and Xba I.

Wild-type and
recombinant A. tumefaciens strains were cultured as described by Zaldivar-Cruz
et al. (2003). In short, single colonies from each bacterial strain were
inoculated into 50-mL Erlenmeyer flasks containing 25 mL liquid YEB medium with
100 mg·L-1 rifampicin, 100 mg·L-1 streptomycin, and either 50 mg·L-1 kanamycin (sulphate salt, Sigma, St.
Louis, Mo.) or, when needed, 50 mg·L-1 hygromycin, all at a pH 5.6. The bacterial cultures were incubated in the
presence or absence of acetosyringone (AS,
3’5’-Dimeth-oxy-4’-hydroxyacetophenone) for 24 hrs at 28ºC on a shaker that was
adjusted to 200 rpm, until an O.D.600nm of 0.6 (late log phase) was
reached.

Plant transformation

At different periods following germination
(2, 4, 6 and 8 weeks), the plantlets were aseptically pre-incubated for different
periods of time (0, 1, 2, 6, 12 and 24 hrs) in liquid MS media without growth
regulators. Then, the A. tumefaciens strains were infiltrated into the
leaves by different methods: (1) The Vacuum method [V]: the leaves were
submerged into the bacterial suspensions and a negative pressure (-1 atm.) was
applied for 15 min; (2) The Infiltration method [I]: the plantlets and the
bacterial suspensions were poured into a 5-mL syringe, then a positive pressure
(+1 atm.) was applied for 5 min; (3) The Micro-injection method [M]: the
bacterial solutions were infiltrated directly, with a needleless syringe, into
different points on the down-side of the leaves; (4) A combination of V and I;
(5) A combination of V and M; (6) A combination of I and M; (7) A combination
of M and I, then V. Following infiltration, each individual explant was rinsed
with sterile MS medium and the co-cultivation was left in the dark for
different periods of time (1, 2 and 3 days) at either 20 or 28ºC. After
co-cultivation, the leaf explants were washed thoroughly with sterile liquid MS
medium and subsequently rinsed four times with 250 mg·L-1 Cefotaxime
(Claforan). Sixty leaf explants were used for each treatment.

Analysis of transformed plants

The efficiency of transient transformation
was evaluated by the histochemical β-glucoronidase
assay, and then by RT-PCR amplification of the transgene transcripts. The leaf
and hypocotyl explants were immersed in X-Gluc solution, consisting of 2 mM
X-Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucoronide), 100
mM Tris-HCl pH 8.0, 50 mM NaCl, 2 mM potassium ferricyanide, and 1% (W/V)
Triton X-100. After 12 hrs incubation at 37ºC in the dark, the explants were
immersed in 70% ethanol until the chlorophyll had been completely bleached.
Then, the explants were photographed under a light
microscope. According to our requirements, only the explants displaying blue colour in the whole leaf area
were considered to be GUS positive. In order to eliminate any possible endogenous GUS activity, the
histochemical GUS assay was previously performed in non-transformed leaf
explants with X-Gluc solutions and with different pH values (4.0, 7.0 and 8.0)
(Alwen et al. 1992). All of the experiments were repeated three times.

For the gene expression analysis, total
RNA was isolated from the transformed leaves using the Trizol® reagent,
according to the manufacturer’s instructions (Invitrogen). RT-PCR was carried out under the standard conditions in a My-Cycler® apparatus (BIO-RAD), with an initial denaturing step at 94ºC for 5 min; cycling conditions were 94ºC for 1
min, 45ºC for 1 min, 72ºC for 1 min; with a final extension of 10 min at 72ºC.
The amplified fragments were fractionated by gel electrophoresis on a 1%
agarose gel, stained with 1 µg·mL-1 ethidium bromide and visualized
under UV light. Amplification of the different transcripts was completed with
the following combination of primers. For the GUS transcript, forward
(5’-AAAGTGTGGGTCAATAAT-3’) and reverse (5’-AAGCCAGTAAAGTAGAAG-3’) primers
amplified a 593 bp fragment. For the PR10 transcript, forward
(5’-TAATGAGTCCACAGTCCCAGTTGC-3’) and reverse (5’-CACCATTTAAGCATAGGCAGAAGG-3’)
primers amplified a 460 bp fragment. For the esterase transcript, forward (5’-CTTCCTGCAGCTTACGACGATG -3’) and reverse (5’- AGCTGCTGCCTTTGATGGTTC -3’)
primers amplified a 560 bp fragment.

To estimate the
difference between the different treatments, a one-way analysis of variance
(ANOVA) was used and the means were compared using the Tukey’s post hoc test. All of the data are reported as the mean ± standard error. For all of the statistical analyses, the percentage
results were transformed to arcsine before the ANOVA. The back-transformed means are presented and the standard
errors are those obtained from the untransformed variables (Zar, 1999). The
statistical significance was preset at P < 0.05, although the P values obtained are indicated. The statistical analyses were done with the
General Linear Model (GLM) module with the Statistica software version 7.0
(StatSoft Inc., Tulsa OK, USA).

Results and Discussion

In our lab, we analyze the interaction
between Phytophthora capsici and the Habanero pepper at physiological,
biochemical and molecular levels. Since one of our research goals is to study
the dispersion of the oomycete mycelia through the leaf explants that are
transformed with defence genes, we wanted to generate a transformation system
that allows homogenous transgene expression through the whole leaf area,
instead of having discrete transformation spots.

In this study, two types of binary vectors
were used, pCAMBIA2301 and pCAMex (Figure 1). The first vector was used
as a control to discriminate the false positives produced by the bacterial
expression of transgenic GUS activity because this vector contains an intron in
the reporter gene that cannot be processed by bacteria. Consequentially, the
GUS staining is attributed to be exclusively from transformed plant tissues.
pCAMex was constructed from pCAMBIA1303 (http://www.cambia.org)
to constitutively express the genes of interest. pCAMex is a versatile binary
vector that, in addition to the classic GUS histochemical detection assay,
allows monitoring of the transformed tissues in vivo through detection
of GFP in a light microscope.

Our first efforts to transform the
Habanero pepper explants transiently, by vacuum infiltrating the bacterial
solutions containing the binary vector controls, yielded no results at all;
instead, the leaf explants showed a low level of GUS background activity, even in
the negative controls. Since members of the Solanaceae family express an
intrinsic β-glucuronidase activity (Plegt and Bino, 1989) that is
dependent on the pH (Sudan et al. 2006), we
assessed whether the endogenous activity in the Habanero pepper could be
eliminated by modifying the pH of the histochemical assay (Alwen et al. 1992).
We incubated the non-treated leaf explants with the GUS staining solution,
buffered at different pH values. We found that incubation of the leaf explants
at pH 4.0 gave a widespread and weak staining that was especially prominent in
the vascular system. The staining was reduced at pH 7.0, and was completely
eliminated at pH 8.0 (Figure 2). These results confirmed that the
Habanero pepper, like other Solanaceae members, possesses an endogenous
pH-dependent β-glucuronidase activity. Therefore, the histochemical
determinations for all of the transformation assays were done at pH 8.0.

Since using a direct application of the
vacuum to infiltrate the bacterial solution gave no positive GUS results, we
applied either a negative or positive pressure, or a combination of both
pressures, in conjunction with a leaf puncture by a syringe needle in order to
infiltrate the bacterial solutions. As can be observed in Table 1, a
combination of the sequential application of a negative and positive pressure
in the punched leaves gave the best results.

It has been
demonstrated that the age of the plant can be a determinant for a tolerance
against pathogenic microorganisms; the older the plant, the more tolerance they
exhibit during an interaction with pathogens (Kim et al. 1989). In addition, several factors are critical for a
successful transformation and these include the use of young seedling explants
(Binns and Thomashow, 1988). Thus, we evaluated whether or not the age of the explants
have had any influence over the transformation efficiency. As can be seen in Figure
3a-b and Figure 4a, the maximum efficiency of transient
transformation with any of the constructs was reached when 4-week-old plantlets
were used as source of explants; with 8-week-old plantlets and older, the
transformation efficiency was compromised and serious tissue necrosis occurred in the explants, although non-homogenous transient transformation was still
observed (Figure 3a).

Acetosyringone (AS), a molecule derived
from wounded plants, is used by A. tumefaciens as a chemo-attractant to
initiate cell adhesion (Binns and Thomashow, 1988). AS is added during the
culturing of bacterial cells to increase virulence and, consequently, the
efficiency of gene transfer can be improved (Sheikholeslam
and Weeks, 1987; Sunilkumar et al.
1999). In our work, there were no significant differences observed between the
different AS concentrations that were evaluated (Figure 4b). However,
there was no homogenous transient transformation through the whole leaf area
observed when AS was not supplied. This absolute AS-dependency has been also
observed in Arabidopsis thaliana (Sheikholeslam and Weeks,
1987) and in C. annuum (Kim et al. 2001b), where AS was one of
the essential components for pepper transformation.

The
co-cultivation was developed for one, two or three days at two temperatures, 20
or 28ºC, and the best results were obtained when the co-cultivation proceeded
for two days at 28ºC (Table 1). The transformation efficiency was
reduced when the co-cultivation occurred for three days, and no results were
obtained when the co-cultivation was executed for one-day at 20ºC. Similar results were reported with C.
annuum by Li et al. (2003) who observed that a co-cultivation period of two
days was optimal for the transformation of pepper cotyledons and hypocotyls,
but a longer (4, 5, 7 days) co-cultivation period resulted in the necrosis of
explants, which impeded shoot regeneration. In addition, in Citrus paradisi the highest number of GUS+ shoots and segments with GUS+ shoots were obtained with a two-day co-cultivation period (Costa et al. 2002).

In order to control the hypersensitive
response of the Habanero pepper leaf explants, induced by co-cultivation with A.
tumefaciens, the effect of explant pre-cultivation with MS medium, without
growth regulators was tested, using the best conditions from the co-cultivation
experiments (2 days in the dark, on a shaker at 200 rpm and at 28ºC). The
one-hour pre-cultivation produced an almost 50% increment of change in the
frequency of the GUS positive leaf explants for all of the constructs (Table
1). However, an extension of the pre-cultivation period (6, 12 and 24 hrs)
did not result in a significant change of the transient transformation (Figure
4c). In the groundnut (Arachis hypogaea), the transformation
efficiency of explants was higher when compared to those transformants that
were obtained without pre-cultivation (Venkatachalam et al. 1998). Li et al.
(2003) believed that the pepper explants, when preconditioned for a relatively
long time, could not be infected well by A. tumefaciens, but that the
preconditioning promoted the transformation of pepper explants. Over two
decades, several studies have presented evidence for the existence of a "window of
competence"; a time during which the cells are susceptible to Agrobacterium transformation, although the basis for this promotion of transformation by a
pre-culturing of the explants has not yet been completely established. It has
been proposed that the plant cells undergo a physiological adaptation to become
competent for transformation during the pre-conditioning step (Sunilkumar et al. 1999; Costa et al. 2002). In
addition, several authors have suggested that the pre-cultivation step has
served to reduce plant-stress (Venkatachalam et al. 1998; Li et al. 2003).

The best results were obtained when the
different factors evaluated were combined, and this produced an efficient
protocol for the transient transformation of the Habanero pepper leaf explants.
The most efficient protocol includes a 1 hr pre-incubation in MS medium without
growth regulators, followed by a
combination of the three transformation methods previously evaluated, addition of 100 µM AS to the YEB medium
during bacterial culturing, and co-cultivation for 2 days in MS sterile liquid
medium without growth regulators, in the dark and on a shaker adjusted to 200
rpm at 28ºC.

We evaluated the expression of transgenes
in the leaf explants that were transformed under these conditions. By means of
RT-PCR assays, high levels of GUS transcripts were detected in the leaves
transformed either with empty vectors or with PR10 and esterase genes (Figure
5a); as expected, GUS transcripts were not detected in the untransformed
leaves (Figure 5a, lane 1). The transcripts for the PR10 and esterase
genes were also detected in the transformed leaves (Figure 5b); however,
both of the genes were also expressed, at lower levels, in the leaves
transformed with the empty vectors (Figure 5b, lanes 3 and 5).
Furthermore, lower levels of PR10 were present in the untransformed controls (Figure
5b, lane 2). As a member of the pathogenesis-related proteins, PR10 may be
expressed as a result of both biotic and abiotic stresses. Esterase is not
classified as a PR-family member, but it is a hydrolase that functions in the
defence against pathogens (Kim et al. 2001a). Thus, it is possible that these
particular genes are expressed as a result of the transformation process
itself, and that they are not the best examples to evaluate when determining
the efficiency of genetic transformation. Nevertheless, as the results of GUS
gene expression are clear, it is perfectly possible that the transformation
procedure resulted in the over-expression of PR10 and esterase genes, in
addition to their basal expression.

Over the past
five years, remarkable progress has been made in the development and
understanding of the A. tumefaciens infection and transformation
processes. The literature reviewed has provided strong evidence that there is a high recalcitrance of the
peppers to the
Agrobacterium-mediated transformation, and to cell culture in vitro. As a consequence, there are no standard
protocols that can be used between the different species, even within the Genus
Capsicum. Researchers around the world use heterologous systems to express the
pepper genes in order to study their biological functions (Kim et al. 2004). In
the present study, a good system for the transient transformation of Capsicum
chinense, the hottest habanero pepper worldwide (Canto-Flick et al. 2008),
has been established. This optimized protocol can thus be used to study pepper gene functions in a
homologous system.